Field of Invention
This invention relates to novel strains of Scheffersomyces stipitis that are useful in converting plant sugars to ethanol.
Description of the Prior Art
An estimated 1.3 billion dry tons of lignocellulosic biomass could be available annually to support ethanol production at a level that would allow the U.S. to reduce its petroleum consumption by 30%. The fibrous, cell-wall material that is characteristic of lignocellulosic feedstocks is difficult to deconstruct and depolymerize into fermentable sugars. The chemical pretreatment required to open the structure of plant biomass to enzymatic hydrolysis results in solutions rich in glucose and xylose, but laden with byproducts that inhibit fermentation, including acetic acid, furfural, hydroxymethyl furfural, and others. Traditional industrial yeasts do not ferment xylose and are not able to survive, grow or ferment in toxic concentrated hydrolyzates which contain sugar concentrations high enough to support the greater than 40 g/L ethanol accumulations needed for economical recovery.
Pichia stipitis is known to ferment D-xylose to ethanol more efficiently than other native yeasts previously described (Prior, et al., Process Biochemistry 24(1), 21-32 (1989)). The Pichia stipitis that had been deposited at the USDA's ARS Culture Collection (deposit accession number NRRL Y-7124) was recently renamed Scheffersomyces stipitis (Kurtzman and Suzuki, Mycoscience 5(2), 2-14 (2010)) and is particularly useful because it has strong NADH-linked, as opposed to NADPH-linked, aldose reductase activity providing for a more favorable cofactor balance in the conversion of xylose to xylulose (Bruinenberg, et al., Applied Micro. and Biotech. 19, 256-260 (1984)). S. stipitis strain NRRL Y-7124 ferments hexoses and xylose to economically recoverable concentrations of ethanol exceeding 40 g/L with almost no accumulation of xylitol byproduct (Slininger, et al., Biotechnology Letters 7, 431-436 (1985); Slininger, et al., Biotechnology and Bioengineering 35, 727-731 (1990a); Slininger, et al., Annals of the New York Academy of Science 589, 25-40 (1990b)). In nutritionally optimized media, this S. stipitis strain is able to produce over 70 g/L ethanol in 40 hours (1.75 g/L/h) from 150 g/L sugars at a yield of 0.41±0.06 g/g and an ethanol productivity of 1.6 g/L/h in high density fermentations (6 g/L cells) (Slininger, et al. (1985); Slininger, et al., Applied Microbiology and Biotechnology 72, 1285-1296 (2006); Slininger, et al., Biotechnology and Bioengineering 108(8), 1801-1815 (2011)). Given appropriate nitrogen levels, it is also relatively resistant to fermentation inhibitors ethanol, furfural, and hydroxymethylfurfural (HMF) (Slininger, et al., Biotechnology and Bioengineering 102(3):778-790 (2009)). Scheffersomyces stipitis is one of the most viable native pentose-fermenting yeasts available for commercial scale-up, as reviewed by Agbogbo and Coward-Kelly (Biotechnology Letters 30, 1515-1524 (2008)) who point to the need to improve sugar uptake rate in biomass hydrolyzates, including reducing the effects of diauxy and improving ethanol and inhibitor tolerance. Thus, there is a need for novel strains of S. stipitis which are tolerant of diverse lignocellulosic hydrolyzates.
To improve the performance of this Scheffersomyces stipitis strain, various adaptation procedures are applied. These adaptation procedures include natural selection on hardwood hemicelluose acid prehydrolyzate (Nigam, J. of Applied Microbiology 90(2), 208-215 (2001a)) and wheat straw hemicelluloses hydrolyzate (Nigam, J. of Biotechnology 87(1), 17-27 (2001b)), UV-C mutagenesis and anaerobic environment selection to reduce oxygen requirement (Hughes, et al., J. of Industrial Microbiology and Biotechnology 39, 163-173 (2012)), and UV mutagenesis followed by genome shuffling and selective plating on hydrolyzate gradient plates to improve fermentation of waste sulfite liquor (Bajwa, et al., Biotechnology and Bioengineering 104, 892-900 (2009); Bajwa, et al., J. of Microbiological Methods 81, 179-186 (2010)). While these experiments suggest the potential utility of an adaptation approach to improving the functionality of Scheffersomyces strains for industrial application on lignocellulosic hydrolyzates, they do not describe the application of adapted strains of S. stipitis to hydrolyzates with sugar concentrations high enough to support economical ethanol production. Thus a need exists to generate adapted S. stipitis that can utilize highly concentrated sugar hydrolyzates so that ethanol production is affordable.